Diagnostics comprise a critical component of defense against select agent infections. The virulence of most bacterial select agents requires rapid detection and early treatment to prevent serious morbidity and mortality. Most bacterial bioterrorism agents cause blood stream infections (BSI) as part of their disease syndrome. Blood-based detection offers the promise of early BSI diagnosis. However, rapid BSI detection is particularly challenging. Assays must be extremely sensitive to detect early disease. The sensitivity required is on the order of < 5 cfu of the select agent per ml of blood. Yet, the diagnostic assays must also be rapid, robust and easy to perform, preferably near the point-of-care. Even the perfect select agent test will be of little use if it is not be available when neede in an emergency. Thus, the best select agent assays must be able to be manufactured upon very short notice, or be very inexpensive, easy to stockpile, and have a prolonged shelf life. The very best assays would have all of these characteristics. Cepheid and the Alland laboratory have a history of successful collaborations devoted to developing rapid, sensitive, on-demand tests for infectious disease. We recently discovered an approach that is likely to provide highly-sensitive BSI detection. Our approach automatically extracts bacteria from 1 - 10 ml of blood and then automatically performs very-small- amplicon nested PCR. The entire test can be performed within a disposable plastic cartridge and a highly- flexible instrument platform. As proof of principal, our preliminary studies detected S. aureus in the blood of almost 90% of S. aureus BSI patients. Here, we propose to further develop our approach to create rapid point-of-care assays that will detect select agent BSIs at the early stage when a patient may be symptomatic but still have low-level bacteremia. We will also develop a strategy that permits rapid manufacture and distribution of appropriate select agent assays within weeks of a bioterrorist attack with or without stockpiling of an initial test lot. Our six specific aims are t: 1) Develop highly-sensitive, small amplicon nested-PCR assays for each category A select agent bacterium and for each unconventional category B bacterial select agent; 2) Develop multiplex assays for major select agent risks; 3) Adapt each assay to the Cepheid cartridge and the GeneXpert(R) platform; 4) Optimize our 1 ml blood processing protocol and develop a method to isolate select agent DNA from 10 ml of blood using Cepheid sample processing cartridges; 5) Perform analytic tests of both uniplex and multiplex assays on simulated clinical samples containing select agents; and 6) Develop an emergency plan for on demand manufacture and emergency FDA use authorization.